A controlled comparison of the efficacy of clomiphene citrate in male infertility*

Transcription

1 FERTILITY AND STERILITY Copyright 1988 The American Fertility Society Printed in U.S.A. A controlled comparison of the efficacy of clomiphene citrate in male infertility* Rebecca Z. Sokol, M.D.t:j: Barbara S. Steiner, R.N., B.S.N. Maria Bustillo, M.D. Gloria Petersen, Ph.D. II Ronald S. Swerdloff, M.D. Division of Endocrinology, Department of Medicine, Harbor-University of California Los Angeles (UCLA) Medical Center, UCLA School of Medicine, Torrance, California To determine whether clomiphene citrate (CC) improves fertility in oligospermic men, 23 men with sperm concentrations between 0.5 and 20 million sperm per milliliter; normal serum gonadotropins and testosterone; and a presumptively fertile partner were enrolled in the study. After a 3-month control period, patients were randomly prescribed CC, 25 mg/day; or placebo, 1 tablet/day, for 12 months. The pregnancy rates for the CC group and the placebo group were 9.09% and 44.44%, respectively (not significant). During the treatment phase, the CC group had significantly higher levels ofluteinizing hormone (LH) serum, follicle-stimulating hormone (FSH), testosterone (T), and estradiol than the placebo group. CC treatment also resulted in greater LH, FSH, and T responses to gonadotropin-releasing hormone (GnRH). There were no differences between the placebo and CC groups for the sperm penetration assay or semen parameters. The authors conclude that CC is not a useful drug in the treatment of male infertility. Fertil Steril 49:865, 1988 Approximately 10% of couples are unable to conceive. Abnormal male reproductive function is believed to be the cause of approximately 50% of the infertility in these couples. Of these men, a majority can be shown to have decreased sperm number. There is no standard established method for the treatment of idiopathic male infertility. Most of the proposed treatment regimens have Received August 24, 1987; revised and accepted January 28, * Supported by the National Institutes of Health grants T32 DK and HD t Recipient of the Clinical Associate Physician Award, National Institutes of Health, General Clinical Research Center, CAP RR :j: Reprint requests: Rebecca Z. Sokol, M.D., Harbor-UCLA Medical Center, 1000 West Carson Street, RB #1, Torrance, California Present address: Genetics and In Vitro Fertilization Institute, Fairfax, Virginia. II Present address: Cedars-Sinai Medical Center, Los Angeles, California. been directed at modifying either the regulating hormones at the hypothalamic-pituitary region and/or altering the intratesticular hormonal milieu. 1 Clomiphene citrate (CC) is one of the most commonly prescribed drugs for the treatment of male infertility. Interest in CC began in the 1960s when a number of studies were published with variable results. The drug usually was administered for 2 to 6 months at a dosage of 25 to 100 mg/day. The dosage was based on the discovery that high doses of CC (200 to 400 mg/day) would suppress spermatogenesis because of its estrogenic effects, whereas doses of 25 to 100 mg would result in an increase in sperm concentrations. 2,3 Some investigators reported increased sperm counts and pregnancies, whereas others reported no improvement with CC The present study was designed to determine in a double-blind controlled prospective manner the relative ability of placebo and CC to increase sperm counts and improve fertility in men with idiopathic oligospermia and infertility. Sokol et al. CC in male infertility 865

2 Research Subjects MATERIALS AND METHODS Twenty-three adult males, ranging in age from 23 to 49 years of age, were enrolled in the study. These men had no evidence of major systemic or psychiatric illness. They carried a diagnosis of infertility, which was defined as an inability to have children after 1 year of regular unprotected intercourse. Criteria for inclusion in the study included: (1) three semen analyses in which sperm count ranged between 0.5 and 20 million sperm/ml, sperm morphology was normal, and sperm motility was> 10%; (2) normal serum values for luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and prolactin (PRL); and (3) a female partner who was determined to be fertile on the basis of history, physical examination, basal body temperature, menstrual history, progesterone (P) levels, and when performed, a normal hysterosalpingogram (18 women) and/or laparoscopy (11 women). Each subject and his partner were informed of the nature of the study and were provided with a written description of the protocol, including any potential adverse effects of the drug and experimental procedures, and the information that one of the drugs to be administered was a placebo. Experimental Design After fulfilling the study criteria, patients were placed into a 3-month control period. The patients then were randomly placed, using a random numbers table, in a double-blind fashion into one oftwo treatment regimens: CC or placebo. The patients were treated for 12 months with either CC (25 mg/ day) or placebo every day. The drug and placebo were dispensed in identical-appearing tablets. There was a follow-up of 12 months. During the control and treatment periods, and during the first 3 months of the follow-up period, semen and blood samples were collected monthly. Semen was evaluated for a standard semen analysis as well as in the human spermatozoa zona-free hamster in vitro sperm penetration assay (SPA). Blood was evaluated for LH, FSH, T, and estradiol (E2). Every 3 months throughout the study, patients underwent a gonadotropin-releasing hormone (GnRH) stimulation test and a physical examination. Semen Analysis Each month, each subject was asked to abstain from ejaculation for 2 days prior to submission of the specimen. Semen sample collections were scheduled to coincide with the partner's menses. The specimens were obtained by masturbation into a clean, dry, wide-mouth glass container and delivered to the examiner within 1 hour of collection. Samples were evaluated in the fashion described by the World Health Organization manual for semen analysis. I6 Sperm Penetration Assay The SPA was performed as previously described. 17,18 After semen analysis, the semen was washed twice in 10 volumes of Biggers, Whitten, and Wittingham's Buffer (BWW) and centrifuged for 6 minutes at 600 X g. The final pellet was resuspended in 1 ml of BWW buffer and diluted with BWW to a final concentration of 1 X 10 7 sperm/ml. Aliquots of 0.5 ml were transferred to sterile falcon tubes with caps (Falcon Plastics 2063, Oxnard, CA). The tubes were placed horizontally in a 95% O2/5% CO2 incubator at 37 C for 17 hours. The hamster ova were obtained by the previously described methods for superovulation ovum recovery and removal of the zona pellucida. 19,2o The average number of eggs analyzed in an assay was 30 ± 3 eggs. A known normal control semen sample was run with each assay. (In our laboratory, fertile male donor sperm penetrates 50 ± 3.0%, range, 34 to 74). All assays were scored by one observer. Hormone Measurement Once a month throughout the control, treatment, and recovery phases, each subject had three samples collected 20 minutes apart for measurement of LH, FSH, T, and E2 by radioimmunoassay (RIA) using techniques long established in the Harbor-UCLA Medical Center, Endocrine Research Laboratories. I9-22 Reagents for LH and FSH were supplied by the National Institute of Arthritis, Digestive Disease and Kidney (NIADDK). Serum T and E2 measurements were performed using antibodies generated in the Harbor-UCLA Medical Center endocrinology laboratories. Each of these methods was specific and sensitive, allowing for measurements of levels below the normal male range. Four large pools of blood containing progressively increasing concentrations of the hormones to be assayed were prepared in advance and 866 Sokol et al. CC in male infertility Fertility and Sterility

3 Table 1 Hormone LH FSH Testosterone Estradiol Hormone Measurements Normal range 2-15 ng/ml 1-15 ng/ml ng/dl pg/ml Intra -assay variation % ±6.6 ±3.3 ±6.7 ±5.7 Interassay variation % ±10.7 ±12.1 ±11.6 ±12.4 median among the conceivers throughout treatment and at their predicted conception dates. The latter values were estimated by linear interpolation between the closest dates on either side of the approximate conception date. Wilcoxon's rank sum test also was used for the analysis. All data also were log-transformed and t-tests were performed; qualitatively identical results were affirmed as with the Wilcoxon tests and are not presented. included in each assay to determine interassay variation and to provide a reference level for each assay. Intra-assay variation was calculated based on duplicate determinations. Normal values and the intra- and interassay variations in our laboratory are listed in Table 1. Blood Collection and Hormone Gonadotropin Releasing Hormone Testing Serum samples were collected prior to and 30, 60, and 90 minutes after a bolus of 100 /lg of GnRH, LH, and FSH responses were measured in the blood samples. Toxicologic Studies Electrolytes, liver and kidney function tests, cholesterol and glucose measurements, and a complete blood count were performed once during the control period, monthly during the treatment period, and once during recovery period. Physical Examination Physical examinations were performed on each volunteer during the screening period and then every 3 months during the control, treatment, and recovery phases of the study to document any side effects and possible changes in physical examination. Statistics To compare the effective treatment on seminal and hormonal parameters, the Wilcoxon's rank sum test was used. For each parameter, three outcomes were analyzed: (1) control period, (2) treatment period, and (3) the differences between the treatment and control periods. To determine whether the parameters could be used to predict conception, approximate dates based on birth dates of the children were used. Analyses were based on the comparison of the median of the parameter for nonconceivers with the Physical Examination RESULTS Side effects noted by patients in the CC-treated group included headaches in one subject and gynecomastia in two subjects. One volunteer receiving CC was noted to have enlarged testes. Ultrasound examination was negative. No significant changes in physical examination were noted in any of the volunteers on placebo. One patient in the placebo group complained of a skin rash on his face. Pregnancy Rates There was one conception in the CC group, for a conception rate of 9.09%, and four pregnancies in the placebo group, for a pregnancy rate of 44.44%. The difference between the pregnancy rates was not significant (P < 0.13). Toxicologic Studies No significant abnormalities were noted throughout the study. Treatment Versus Placebo Groups The results for the differences for semen parameters, SPAs, and hormonal values between treatment and placebo groups are noted in Tables 2 and 3. During the control period, there were no significant differences in the SPA results, sperm analyses, or hormonal values between the two groups. During the treatment phase, there were no differences between the two groups for SPA results or seminal parameter results. There were significant increases in LH and FSH levels in the CC treatment group compared with the control group (P < 0.01; P < 0.02). Serum T and E2 were higher in the CC-treated group as compared to the placebo group (P < 0.01), as were the stimulated responses of LH, FSH, and T to GnRH stimulation testing (P < 0.01). There were no significant differences in Sokol et al. CC in male infertility 867

4 Table 2 Clinical Trial on the Effect of Clomiphene Citrate on Oligospermic Men a Treatment group = placebo Variable n Median Mean deviation Sperm count/ml (X10 6 ) Sperm motility (%) Total sperm count (X10 6 ) Sperm penetration (%) E2 (pg/ml) LH-O' (ng/ml) LH-90' (ng/ml) FSH-O' (ng/ml) FSH-90' (ng/ml) T-O' (ng/dl) T-90' (ng/dl) Treatment group = clomiphene P values t-test n Median Mean deviation Wilcoxon Logs a Summary statistics for baseline treatment versus placebo groups. semen or hormone factors between conceivers and nonconceivers. DISCUSSION CC is one of the most commonly used drugs in the treatment of male infertility. In low doses, CC acts as a competitive inhibitor of estrogen action. In high doses, it has mild estrogenic activity. It is theorized that this drug exerts its effects by two mechanisms of action. One hypothesis is that the feedback of androgens at;he hypothalamic level is modified by peripheral and hypothalamic aromatization of T to E2. E2 then binds to hypothalamic receptors and inhibits gonadotropin production. Based on this theory, it is postulated that antiestrogens will displace estrogens from their receptors and therefore interfere with the normal feedback regulation by androgenic and estrogenic steroids. This interference results in increased secretion of GnRH which, in turn, stimulates gonadotropins to increase T production and germinal cell maturation. 1,23 Our results indicate the CC does indeed interfere with the feedback of androgens at the hypothalamic and/or pituitary levels, resulting in increased circulating levels of T and E 2, because our data demonstrate that pituitary responsiveness to GnRH is increased by CC treatment. The predominant effect appears to be on the hypothalamus. Furthermore, our data are not in agreement with the other postulated mechanism of action of antiestrogens, which theorizes that drugs such as CC have a direct effect on the testis by interfering with Leydig cell function. 1 In this latter schema, the ad- Table 3 Clinical Trial on the Effect of Clomiphene Citrate on Oligospermic Men a Treatment group = placebo Treatment group = clomiphene P values t test Variable n Median Mean deviation n Median Mean deviation Wilcoxon Logs Sperm count/ml (X10 6 ) , Sperm motility (%) ,24 Total sperm count (X10 6 ) Sperm penetration (%) E2 (pg/ml) LH-O (ng/ml) LH-90' (ng/ml) FSH-O' (ng/ml) FSH-90' (ng/ml) T-O' (ng/dl) T-90' (ng/dl) a Summary statistics for change from baseline to mean over treatment period (treatment versus placebo groups). 868 Sokol et al. CC in male infertility Fertility and Sterility

5 ministration of an anti -estrogen is thought to decrease the biologic effectiveness of intratesticular E2 and therefore lead to an increase in sperm production by the testis in oligospermic men. Our data indicate that there was no significant preferential increase in sperm concentration or total sperm count in those patients who were treated with CC as compared with their normal controls. Indeed, from initiation into the study until its completion, all sperm counts increased with time, regardless of whether the patient was receiving the placebo or CC. Similar results have been observed by others. 24,25 It is theorized that the patient who is infertile is seen at tertiary referral centers at a time when his sperm count is at its lowest ebb; and with time, the sperm count naturally increases back to a more normal level. Results such as these emphasize the importance of placebo-controlled trials. Our data also indicate that it is highly unlikely that CC is an efficacious drug in the treatment of male infertility and oligospermia. We had more pregnancies in the placebo treatment group then we did in the CC treatment group. Five previous studies dealing with the use of CC as a treatment for oligospermia have included controls. Weiland et aly administered CC, 5 or 10 mg per day for 10 weeks, followed by a placebo course of equal duration. They reported that sperm counts increased in an erratic manner and no pregnancies occurred. Masala et al. 12 administered CC, 50 mg twice a day for 5 days, to 10 oligospermic men and 10 controls. No significant differences were detected in gonadotropin and T levels between the two groups. Changes in sperm counts were not reported. The study conducted by Foss et al. 13 is unique in that they studied 114 men, approximately five times as many subjects as any of the other studies. In this controlled double-blind study, the patients received either 100 mg/day of CC for 10 days each month for 3 months, or a placebo. Although 19 pregnancies occurred, they were unable to implicate CC as the effective agent. More recently, Wang and co-workers l4 from the University of Hong Kong reported on the relative effectiveness of several putative therapeutic regimens for oligospermic men. These included placebo, CC, mesterolone, pentoxifylline, and T rebound therapy. Forty-six men with idiopathic oligospermia were randomly assigned to treatment groups that ranged in size from 6 to 18 subjects. Most treatment regimens were for 6 months. Pregnancy rates of 36.4% (4 out of 11) and 22.2% (3 out of 18) were observed in partners of men receiving CC 25 mg/ day and 50 mg/day, respectively. No pregnancies were noted in the partners of the men receiving the other regimens. In this group, as in our group, there were no significant differences in SPA results while on CC treatment. 15 Unlike our study, these investigators did not evaluate the placebo-treated group with sperm penetration testing. We conclude that CC is not a useful drug in the treatment of male infertility. Although our numbers of pregnancy are small, we were unable to identify any predictable factors for those patients who were to become pregnant as compared with those who were unable to impregnate their spouses. The results of our study also reiterate the necessity for inclusion of placebo-treated subjects into therapeutic trials. Without a placebo group, the semen data, in combination with a pregnancy in the CCtreated group, may have been interpreted as "suggestive" of a positive role for CC in the treatment of male infertility. The inclusion of a placebo arm in the study excludes this possibility. Acknowledgments. We acknowledge the excellent technical assistance provided by Ms. Margaret Peterson, Mr. Brad Brunskill, Ms. Carol Madding, and Ms. Helen Okuda. We also thank Mrs. Phyllis Flemming and Ms. P. Maddy Daly for their expert secretarial skills. REFERENCES 1. Sokol RZ, Swerdloff RS: Male infertility: diagnosis and medical management. In Infertility: Diagnosis and Management, Edited by J Aiman. New York, Springer-Verlag, 1984, p Heller CG, Rowley MH, Heller GV: Clomiphene citrate: a correlation of its effects on sperm concentration and morphology, total gonadotrophin and testicular cytology in normal men. J Clin Endocrinol Metab 29:638, Paulson DF: Endocrine therapy of male infertility, with special reference to clomiphine citrate (Abstr). Fertil Steril 28:329, Jungck EC, Roy S, Greenblatt RB, Mahesh VB: Effect of clomiphene citrate in spermatogenesis in the human: a preliminary report. Fertil Steril 15:40, Mellinger RC, Thompson RJ: The effect of clomophine citrate in male infertility. 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